The focus of this application is on elucidating the molecular mechanisms by which transglutaminase 2 (TG2) regulates hypoxia inducible factor (HIF) signaling and attenuates ischemic-induced cell death. In response to ischemia there is an upregulation of genes that can facilitate either cell survival or cell death. Whether cell survival or delayed cell death is the final outcome is dependent on the complement of ischemia-induced genes expressed, which can differ depending on variables such as duration and severity of the insult and the presence of regulatory proteins. HIF, which is composed of the oxygen sensitive HIF1 subunit and the constitutively expressed HIF12, is the transcription factor that is primarily responsible for the upregulation of hypoxic responsive genes. We have demonstrated that TG2 binds HIF12, attenuates HIF signaling, attenuates the expression of specific HIF- responsive genes and protects against ischemic insult. Furthermore, we have compelling data demonstrating that neuronal expression of human TG2 significantly reduces infarct volume in a mouse stroke model. The central hypothesis of this application is that TG2 is a regulator of hypoxic-induced transcriptional signaling, and that TG2 plays a fundamental role in protecting neurons against ischemia-induced cell death. The objectives of this proposal will be met through three specific aims that test the hypotheses: (1) that the TG2- mediated suppression of HIF signaling is dependent on the localization of TG2 to the nucleus, independent of transamidating activity and requires interaction with HIF12, (2) that the protective effects of TG2 against ischemic-induced cell death are independent of transamidating activity, but require nuclear localization and further, that the interaction of TG2 with HIF1 is necessary for the protective role of TG2 against ischemic insult, and (3) that TG2 attenuates the delayed cell death that occurs in response to stroke in vivo. Overall, this is an integrated, mechanistic and holistic proposal. In our studies we will be using recombinant proteins to define the interacting domains between TG2 and HIF1, cell models to understand how TG2 modulates HIF dependent transcriptional events and attenuates ischemia-induced cell death, and mouse models to delineate the role of neuronal TG2 in decreasing stroke damage. We believe that by using this full complement of approaches we will be able to more completely understand the role of TG2 in ameliorating ischemia-induced cell death. These are exciting and innovative studies in that they not only define a new framework for understanding the function of TG2, but that they also are providing new insights into the regulation of ischemia-induced cell death processes.